Complex post-translational regulation of the plant shikimate pathway

Post-translational modifications (PTMs) are crucial regulators of protein function, influencing most cellular processes in both prokaryotes and eukaryotes. The fundamental effect of PTMs on metabolic control has been reported for most metabolic pathways in organisms ranging from animals and plants to bacteria and fungi. However, the impact of PTMs on the regulation of the plant shikimate pathway, which connects central carbon metabolism with the synthesis of aromatic amino acids and represents one of the largest metabolic fluxes on Earth, remains largely unexplored. In this study, we address the identification of PTMs affecting the enzymes of the shikimate pathway in Arabidopsis thaliana. Following a strategy based on transient expression in protoplasts, immunoprecipitation and mass spectrometric analysis, we have identified a large and varied collection of PTMs affecting the 20 alternative isoenzymes involved in the shikimate and phenylalanine (Phe) biosynthesis pathways. Our analysis revealed glycosylation, phosphorylation, methylation, nitration, and acetylation as the predominant modifications. Notably, we have identified multiple phosphorylation events that expand those previously reported. Additionally, we characterized two phosphorylation events affecting chorismate mutase 1 (AtCM1), the committed enzyme in the phenylalanine/tyrosine biosynthetic pathway. Enzymatic assays using phosphomimic recombinant pure AtCM1 support that these PTMs significantly modulate the enzyme´s allosteric behaviour, highlighting its PTMs role in fine-tuning metabolic regulation. Furthermore, functional characterization of prephenate aminotransferase (PAT) showed that phosphomimetic substitutions at Ser240 and Ser247 abolished enzymatic activity, supporting a regulatory role for phosphorylation in aromatic amino acid biosynthesis. Overall, the identification of these PTMs provides new insights into the regulation of metabolic flux through the shikimate and Phe biosynthetic pathways.